Directed molecular evolution of enzymes is a developing field in the biotechnology industry and occurs through the single or repeated application of two steps: diversity/library generation followed by screening or selecting for function. The last several years have produced much progress in each of these areas. Techniques of diversity generation in the creation of libraries range from methods with no structure/function prejudice (error-prone PCR; mutator strains) to highly focused randomization based on structural information (site-directed mutagenesis; cassette mutagenesis). DNA recombination (DNA-shuffling, StEP, SCRATCHY, RACHITT, RDA-PCR) requires no structural information but works on the premise that Nature has already solved the problem of creating functional proteins from amino acids. By randomly recombining the genes for related proteins, new combinations of the different solutions are created which may be better than any of the original individual proteins. Structure-based approaches can be combined with other methods to generate greater diversity.
Advances have also been made in screening the generated libraries for proteins with desired properties. In a screen each protein in the library is analyzed for function, which limits library size. In contrast, genetic selection evaluates entire libraries at once, in a highly parallel fashion, because only functional members of the library survive the selective pressure. In selection, nonfunctional members of the library are not individually evaluated. For screens, each variant must be individually assayed and the data evaluated, requiring more time and materials. In vivo genetic selection strategies enable the exhaustive analysis of protein libraries with up to about 1010 different members. The quoted throughputs are maximal values for industrial, robot driven laboratories. Realistically, experience indicates that an academic, individual investigator laboratory can achieve up to 104 samples/day for screening in yeast and 107 samples/day for genetic selection in yeast. In summary, genetic selection is generally preferable to screening not only because it is higher throughput, but also because it requires less time and materials.
With regard to selection, there are several common conventional selection strategies, such as (i) antibiotic resistance, (ii) substrate selected growth, where degradation of substrates provides elements essential for growth (such as C, N, P, and S), iii) auxotrophic complementation to restore metabolic function, and iv) phage display, which displays peptides or proteins on a virus surface and segregates them on the basis of binding affinity. Although powerful, these selection strategies are not general enough to apply to engineering enzymes for many interesting reactions. Conventional systems rely on screening techniques rather than selection techniques because selections are more difficult.
The generation of libraries has spawned many companies, in fact, spawned an industry. What has so far failed to be addressed is a general method of evaluating libraries (no matter how they are generated) through genetic selection. Accordingly, there is a need for new compositions and methods for engineering polypeptides and rapidly identifying engineered polypeptides having desirable characteristics.